A common procedure for the chemical synthesis of nucleic acids involves step-wise assembly of suitable, protected phosphoramidite building blocks on solid phase, followed by the removal of protecting groups and the detachment from the solid phase. In addition to the incorporation of ribo- or deoxyribonucleotides, this procedure allows the introduction of other, additional compounds including nucleotide analogs (containing modified sugar moieties and/or modified nucleobase moieties) as well as other molecules which facilitate specific detection and characterization (e.g., dyes) or specific binding (e.g., biotin). The introduction of these compounds into nucleic acids requires the presence of reactive groups and protecting groups that are compatible with the assembly and deprotection protocols employed in the assembly and deprotection of nucleic acids. Ideally, these additional compounds contain a removable reporter/protection group, which allows the in-line monitoring of the coupling efficiency.
The named modifications can only be introduced into nucleic acids if they are stable, both under assembly and deprotection conditions. A variety of very useful and routinely employed dyes and reporter groups are not sufficiently stable and are therefore introduced only after assembly and deprotection of the nucleic acids by so-called conjugation reactions. For their selective and efficient conjugation, an unfunctionalized, reactive, and stable functionality is introduced into the nucleic acid during assembly. The reactive functionalities are usually amino- or thiol-groups. The corresponding building blocks, usually called “amino linkers” or “thiol linkers,” respectively, contain typically a phosphoramidite moiety for the attachment to the nucleic acid, a linker chain (alkyl-chains or oligoethylenglycol-chains of various lengths) and the suitably protected reactive amino group or thiol group, respectively.
The reactive amino groups of amino linker building blocks presently known are protected either by a N-trifluoracetyl-group (Formula I) or by a N-monomethoxytrityl-group (Formula II). Phosphoramidite derivatives of amino linkers containing a primary amino group which is protected as a trifluoracetyl-derivative (Formula I) can be attached to the 5′-end of a nucleic acid in a usual procedure; after coupling, capping, and oxidation, the still protected amino linker is connected via a phosphoric acid triester moiety to the nucleic acid. The trifluoroacetamido protecting group is then cleaved together with the protecting groups of the nucleobase and the phosphodiester upon treatment of the product with ammonia or methylamine.

N-Trifluoroacetyl-protected amino linkers can be prepared in a simple sequence of reactions from cheap compounds, and their deprotection is very straightforward. Unfortunately, their protecting group cannot be used for controlling coupling efficiency, and neither “trityl-on” purification nor solid support functionalization/derivatization reactions are possible.
Phosphoramidite derivatives of amino linkers comprising a primary amino group which is protected by a monomethoxytrityl group (Formula II) are also attached to a nucleic acid according to the usual procedure, and after capping and oxidation, the monomethoxytrityl protecting group is cleaved under (acidic) detritylation conditions, while the nucleic acid sequence is still attached on the solid support. These formula II monomethoxytrityl-protected amino linkers presented in formula II provide the advantage of simple preparation while allowing for “trityl-on” purification and solid support functionalization/derivatization. Such Formula II amino linkers, however, allow only qualitative control of coupling efficiency and are not completely stable during storage and incorporation into nucleic acids. It is, therefore, desirable to provide an amino or thiol linker building block which is easily produced and stable and which allows control of coupling efficiency, “trityl-on” purification, and solid support functionalization/derivatization.